While non-typhoidal Salmonella (NTS) have long been known to cause gastroenteritis, multiple antibioticresistant highly virulent strains are emerging as important causes of invasive bacteremia and focal infections in the USA and globally, resulting in hospitalizations and deaths. This translational research proposal tests the hypothesis that appropriately engineered attenuated strains of Salmonella enterica serovar Typhimurium and Enteritidis, with attenuating mutations in guaBA (encoding guanine nucleotide biosynthesis enzymes) and either dpX (encoding ATPase) or dpP (encoding a protease), can play an important role in vaccinating against these NTS serovars by: 1) allowing safer, high yield preparation of purified O polysaccharide (OPS) and flagella protein for making conjugate vaccines (dpP and c/pX mutants hyper-express flagella), and 2) by their use in a heterologous mucosal prime/parenteral boost immunization strategy in which mice given the attenuated strains of S. Typhimurium and S. Enteritidis orally are subsequently boosted parenterally with conjugate vaccines consisting of Salmonella Group B and D OPS covalently linked to Phase 1 flagella protein of Typhimurium or Enteritidis, respectively. We hypothesize that this strategy will markedly broaden the immune responses elicited and enhance protection (tested in oral challenge studies in mice) over what can be achieved with either oral vaccines or conjugates alone. We expect SlgA antibodies and cellmediated immunity (CMI) stimulated by the live vaccine to synergize with the serum IgG anti-OPS bactericidal antibodies and anti-flagella antibodies stimulated by the parenteral conjugate vaccines. Two S. Paratyphi A strains, genotypes guaBAc/pXand guaBA.dpP, that have already been constructed, will be fed to volunteers in a Phase 1 clinical trial in grant-year 1 to obtain a preliminary assessment of these attenuations in humans (albeit in Paratyphi A background) and their likely suitability for attenuating NTS. Since Typhimurium and Enteritidis are the most common NTS serovars associated with invasive and severe gastrointestinal NTS clinical disease in the USA (and globally), this research can pave the way for development of a rational, highly effective, broad spectrum vaccine against NTS. If the bivalent vaccines cross protect against other Group B &D serovars and if (in the future) either a group C1 or C2 conjugate is added, coverage will then be provided against the overwhelming majority of NTS associated with invasive and severe disease in the USA and worldwide.